This invention relates to a device for carrying out a procedure, for example an analytical procedure, in which microorganisms or other biological material interacts with components of a fluid.
Microorganisms are employed, because of their wide variety of metabolic pathways, for investigations into the biodegradability of various substances, for analytical purposes, for example for assaying individual substances or classes of substances, and for assessing the extent of pollution of water with biodegradable substances.
Frequently these procedures entail determining the metabolic activity of the microorganisms using suitable sensors. Thus the metabolic activity increases when the sample to be investigated contains substances which are used by the microorganisms as nutrients. Thus, biological oxygen demand (BOD), which is used as indicator for water pollution, is based on determination of oxygen consumption through the metabolic activity of microorganisms which are incubated with the sample to be investigated.
Microorganisms can also be used as toxicity indicators, because the metabolic activity decreases in the presence of toxic substances which lead to a reduction in the activity or even to death of the cells.
Microorganisms can in the above applications be employed as conventional cultures in minireactors or shake cultures in order to follow the metabolic activity of the cells, for example on the basis of the oxygen consumption or the cell growth, over a lengthy period. However, for some years there have also been approaches to shortening the analysis times required by automating sample delivery and shortening the incubation time, especially when it is not necessary to achieve an equilibrium state. In addition, microorganisms can be combined directly with the detectors by enclosing the microbial cells in polymers directly on electrodes or between membranes. Also in some circumstances polymers may be used in which the organisms are embedded for additional protection. These membranes may be fixed adjacent to a detector, for example an electrode with special mounting devices.
Microbial sensors, for example for determining a BOD value, may be fabricated in this way. However, if these sensors are used as detectors in automated analytical systems, special forms of construction need to be provided on the microbial membrane to enable the sample to be passed over the membrane. As a rule, only a small part of the membrane then comes into contact with the sample and for only a short time. The active surface of the membrane, which is restricted by the size of the detector and sample delivery construction, has a limiting effect, as a result of which only a restricted amount of microorganisms can be actively utilized.
Other forms of construction consist of cartridges (known from the use of immobilized enzymes) which are filled, as small columns, with polymers or glass particles on which enzymes or other proteins can be immobilized. Compared with membranes, they have the advantage that the amount of proteins which can be actively utilized is limited only by the size of the cartridge. In addition, because the sample flows through the cartridges of this type, a good contact between proteins and sample is ensured, and thus a good conversion of the analyte is achieved, which leads to relatively large signals. Cartridges of this type are used in flow systems in which the detector is then located downstream.
It is possible in principle to enclose microorganisms in cartridges of this type, because microorganisms can be adsorbed onto porous carriers. However, flow through the reactors is successful only when the internal pressure in the cartridge is not too high. The internal pressure depends, on the one hand, on the particles used to adsorb the cells and, on the other hand, on the mesh width of the gratings used to retain the particles in the cartridge. The fact that the cells are merely adsorbed onto the carrier particles means that they can also be washed off. If the mesh width of the gratings is so small that not only the particles but also washed-off microorganisms are retained in the cartridge, then the internal pressure becomes so high that conventional peristaltic pumps and, even more so, micropumps can no longer ensure transport of the sample through the cartridges. However, without appropriate membranes the microorganisms are discharged from the system, which can lead to falsification of the signals. An additional factor is that in cartridges of this type, reproducible cell loading can be achieved only with difficulty because the number of cells adsorbed on carriers cannot be easily regulated.
The invention is therefore based on the object of developing a novel device for contacting microorganisms or other biological material with fluids, e.g. liquids to be analyzed. The invention particularly relates to microbial membrane reactors for use in a flow system, in which microorganisms can be introduced easily and reproducibly, and discharge of the organisms from the reactor is prevented.
It will be appreciated that where in the following description, microorganisms are referred to, other biological materials may be employed instead of microorganism as such. Thus the term xe2x80x9cmicroorganismxe2x80x9d is intended to embrace not only prokaryotic and eukaryotic unicellular organisms but also cells or tissues of human, plant or animal origin.
Thus where the term xe2x80x9cmicrobial membrane reactorxe2x80x9d is used, it is intended to refer to a device including a membrane provided with any of the forms of xe2x80x9cmicroorganismxe2x80x9d encompassed by the above definition.
According to the present invention there is provided a device for carrying out a process in which microorganisms or other biological materials interact with components of a fluid, comprising a membrane sandwiched between opposed surfaces of first and second structural elements, characterised in that the microorganisms or other biological materials are located between the first structural element and the membrane and the inner surface of the second structural element that abuts the membrane is provided with one or more flow channels for flow of said fluid over the surface of the membrane. The membrane, which is preferably sterile, may be permeable to the fluid and substances dissolved in the fluid, e.g. dissolved gases and substances that may be metabolised by or otherwise interact with the microorganisms.
The present invention paarticularly relates to a microbial membrane reactor for use in flow systems, which has a first element with a planar inner surface to receive the microorganisms and a second element which is likewise provided with a planar inner surface and on which at least one flow pathway is formed, where the first and second element are arranged with their insides in contact, and a sterile membrane is arranged between the elements.
The pore size of the sterile membrane is preferably chosen so that it is impervious to the microorganisms used. The microorganisms are preferably arranged in a microbial membrane located between the inner surface of the first element and the sterile membrane. In other words, the sterile membrane covers the microbial membrane in relation to the flow pathways.
The invention also includes reactors which do not use microbial membrane. This may be by, for example, directly applying the microorganisms to the side of the sterile membrane facing the first element. This might be achieved by filtering the cells through the sterile membrane. It is also possible furthermore for the microorganisms to be applied directly to the planar inside of the first element, for example by applying a solution, a gel or a paste etc., comprising the microorganisms.
The elements of the membrane reactor can preferably be connected together by fixing means, for example by screws which grip through appropriate passages bored in the elements so that the elements are pressed together. However, other connection techniques such as gluing or bonding are possible.
The flow pathways are preferably designed as flow channels, it being possible to alter the contact area between the flow channel and the microorganisms present in the microbial membrane through the dimensions of the flow channel and its geometry.
In addition, connecting bores at the start and end of each flow channel lead to the upper side of the second element in order to be able to connect the liquid flow to the flow channel and thus define the flow pathway.
The flow channels are preferably formed in the inner surface of the substrate of the second element, for example by etching, cutting or another material-removing technique. However, the flow pathways can also be formed in a sheet which is applied to the planar inner surface of the second element, in which case the second element has corresponding connecting channels to connect the flow pathways of the sheet to the liquid flow. The flow channels can also be produced by material-adding processes on the planar inside of the second element, which then assumes the function of a substrate. A structural coating of this type would be, for example, the application of a paste by a screen printing technique with subsequent curing by drying or baking etc. Processes of these types are known to the skilled person from thick film technology and therefore do not need to be explained in detail here.
The microorganisms are preferably located on or in the microbial membrane which is placed on the inner surface of the first element. This may take the form of filter paper through which a cell suspension has been filtered. The number of cells adsorbed on the filter paper depends on the number of cells in the suspension. This can easily be assessed via the turbidity of the suspension, that is to say its optical density, and can thus also be more easily adjusted reproducibly than with the membrane or cartridge reactors of the prior art.
The microbial membrane may be covered in relation to the flow pathway or the flow channels with a membrane which has pores sufficiently fine for the microorganisms not to be discharged from the system, but so that nutrients and other substances which can be degraded are able to pass through. Since the liquid flow does not flow through the membranes but, because of the construction of the channels or of the sheet, only flows past them, these membranes do not lead to an increased buildup of pressure in the system.
It is thus possible with this type of reactor for the numbers of cells not only to be adjusted very reproducibly but also for the number of cells not to change due to discharge from the system. In contrast to cartridge reactors, the buildup of pressure through the reactor is very much less because no particles are present in the membrane reactor. This means that this type of reactor is also suitable for combining with micropumps with only low capacities. In addition, the contact area between the flow pathway and the microorganisms is very much larger and can easily be altered through the dimension of the flow channel, that is to say its diameter, length or the geometric shape. This means that effective utilization of the enclosed cells is possible, even if there is a system-related change in the limiting conditions. It is furthermore possible easily to manufacture multichannel reactors which are advantageous for ensuring longer operating times and for shortening the time between successive analysis steps. This is because the cells after having been incubated with a sample need to recover from this pollution and to be subjected to rinsing steps. These rinsing steps frequently limit the frequency with which successive analysis procedures can be carried out. It is possible on use of a multichannel system for the incubation with the sample to take place in one channel, while the regeneration of the cells takes place in another channel.
The devices provided according to the invention therefore have the following advantages over the prior art:
the microorganisms may be located on a membrane which is covered with a membrane acting as sterile barrier. This prevents discharge of cells from the reactor.
the microorganisms can be applied to the xe2x80x9cmicrobialxe2x80x9d membrane by filtration of a cell suspension, which makes it possible easily to vary the cell loading and, at the same time, makes good reproducibility of cell loading possible.
the reactor contains no particles to which microorganisms are applied, so that no additional internal pressure is built up.
it is possible by varying the geometry of the flow pathway for the contact time between the sample and the cells to be varied and adapted to practical requirements.
the reactor can be manufactured in various ways so that, for example, combinations with microsystems are easily possible.
compact integration of a plurality of channels is possible.
It is furthermore possible to arrange necessary sensors downstream from the reactor, or they are placed in front of and behind the reactor for differential measurement. Examples of suitable sensors are oxygen electrodes, but other sensors can also be employed depending on the analyte to be determined and microorganisms used, such as pH, CO2, NH3 or Clxe2x88x92 electrodes. The selection of the detectors depends on the particular requirements of the analysis procedures being carried out.